Blocking IL-9 signaling in conjunction with chemotherapy to treat cancer

ABSTRACT

This document provides methods and materials related to using inhibitors of IL-9 signaling in conjunction with chemotherapy to treat cancer (e.g., solid tumors). For example, methods and materials for using inhibitors of IL-9 signaling (e.g., anti-IL9 antibody preparations) in conjunction with chemotherapy to treat cancer (e.g., solid tumors such as breast cancer tumors or colon cancer tumors) or to reduce the growth rate of cancer (e.g., solid tumors such as breast cancer tumors or colon cancer tumors) within a mammal are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/682,493, filed Apr. 9, 2015 (now U.S. Pat. No. 9,833,512), whichclaims the benefit of U.S. Provisional Application Ser. No. 61/977,558,filed Apr. 9, 2014. The disclosures of the prior applications areconsidered part of (and are incorporated by reference in) the disclosureof this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA155295 awardedby National Institutes of Health. The government has certain rights inthe invention.

BACKGROUND 1. Technical Field

This document provides methods and materials related to using inhibitorsof IL-9 signaling in conjunction with chemotherapy to treat cancer(e.g., solid tumors). For example, this document provides methods andmaterials for using inhibitors of IL-9 signaling (e.g., anti-IL9antibody preparations) in conjunction with chemotherapy to treat cancer(e.g., solid tumors) or to reduce the growth rate of cancer (e.g., solidtumors) within a mammal.

2. Background Information

IL-9 is a paradoxical cytokine, as it mediates both pro-inflammatoryevents and tolerance induction. It is secreted by a host ofpro-inflammatory immune cells including Th9 cells (Stassen et al., Ann.NY Acad Sci., 1247:56-68 (2012)), Th17 cells (Nowak and Noelle,Immunology, 131:169-73 (2010), CD8⁺ Tc9 cells (Visekruna et al., Eur. J.Immunol., 43:606-18 (2013)), eosinophils, mast cells, and innatelymphoid cells (Stassen et al., Ann. NY Acad Sci., 1247:56-68 (2012),Goswami and Kaplan, J. Immunol., 186:3283-8 (2011), Jabeen and Kaplan,Curr. Opin. Immunol., 24:303-7 (2012), Noelle and Nowak, Nat. Rev.Immunol., 10:683-7 (2010), and Schmitt and Bopp, J. Clin. Invest.,122:3857-9 (2012)). It also is associated with tolerogenic cells such asT regulatory cells (Tregs). In this population IL-9 enhances Tregsuppressive potency in an autocrine fashion (Elyaman et al., Proc. Natl.Acad. Sci. USA, 106:12885-90 (2009)), while promoting T cell tolerancevia a paracrine impact upon mast cells (Eller et al., J. Immunol.,186:83-91 (2011), Lu et al., Nature, 442:997-1002 (2006), and Yang etal., PLoS ONE, 5:e8922 (2010)). This wide range of action is followed byan equally wide range of pathologies involving IL-9 secretion.

Most commonly IL-9 is linked to Th2 responses such as parasite expulsionand allergic airway inflammation, but it is also involved inautoimmunity and graft-versus-host disease (Noelle and Nowak, Nat. Rev.Immunol., 10:683-7 (2010)). IL-9 can be secreted by cells that promoteopposite ends of the immune spectrum. For example: pro-inflammatory Th17cells can produce IL-9 and exacerbate experimental autoimmuneencephalitis (EAE) (Nowak et al., J. Exp. Med., 206:1653-60 (2009),whereas IL-9 secreted by Tregs renders them more suppressive andprotects against EAE (Elyaman et al., Proc. Natl. Acad. Sci. USA,106:12885-90 (2009)).

IL-9 also has seemingly contradictory roles in tumor biology. In somehematological tumors, the presence of IL-9 contributes to theestablishment of a tolerogenic/immunosuppressive environment, or actsdirectly to drive tumor growth. IL-9 promotes the proliferation orsurvival of human lymphoid tumors such as Hodgkins lymphoma, acutelymphoblastic leukemia, myeloid leukemia, diffuse large B cell lymphoma,and NK T cell lymphoma (Merz et al., Blood, 78:1311-7 (1991), Lemoli etal., Blood, 87:3852-9 (1996), Lemoli et al., Leuk. Lymphoma, 26:563-73(1997), Lv et al., Int. J. Clin. Exp. Pathol., 6:911-6 (2013), Lv andWang, Leuk. Lymphoma, 54:1367-72 (2013) and Nagato et al., Clin. CancerRes., 11:8250-7 (2005)). It also promotes the proliferation, migrationand adhesion of human lung cancer cells (Matsushita et al., Leuk. Res.,21:211-6 (1997)).

IL-9, however, exhibits the opposite effect on melanoma biology in thatit inhibits growth of B 16 melanoma seeding in the lungs (Purwar et al.,Nat. Med. 18:1248-53 (2012) and Lu et al., J. Clin. Invest., 122:4160-71(2012)), through its effect directly on the lung epithelium, which thenrecruits dendritic cells.

SUMMARY

This document provides methods and materials related to using inhibitorsof IL-9 signaling in conjunction with chemotherapy to treat cancer(e.g., solid tumors). For example, this document provides methods andmaterials for using inhibitors of IL-9 signaling (e.g., anti-IL9antibody preparations) in conjunction with chemotherapy to treat cancer(e.g., solid tumors such as breast cancer tumors or colon cancer tumors)or to reduce the growth rate of cancer (e.g., solid tumors such asbreast cancer tumors or colon cancer tumors) within a mammal. In somecases, inhibitors of IL-9 signaling (e.g., anti-IL9 antibodypreparations) can be used alone or in conjunction with chemotherapyand/or anti-cancer immunotherapy to treat cancer (e.g., solid tumors) orto reduce the growth rate of cancer (e.g., solid tumors) within amammal. For example, inhibitors of IL-9 signaling (e.g., an anti-IL-9antibody preparation) can be used to treat a mammal suffering frombreast cancer or colon cancer.

In general, one aspect of this document features a method for treating amammal having colon or breast cancer. The method comprises, or consistsessentially of, administering a chemotherapeutic agent and an anti-IL-9or anti-IL-9 receptor antibody preparation to the mammal underconditions wherein the progression of the colon or breast cancer or thenumber of colon or breast cancer cells within the mammal is reduced. Themammal can be a human. The method can comprise administering thechemotherapeutic agent to the mammal before the preparation isadministered to the mammal. The method can comprise administering thechemotherapeutic agent to the mammal at least one month before thepreparation is administered to the mammal. The method can compriseadministering the chemotherapeutic agent to the mammal at least twomonth before the preparation is administered to the mammal.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-I. TUBO and 4T1 mammary carcinomas are rejected in IL-9ko mice.Growth of 1×10⁶ TUBO cells implanted s.c. in the flank of (A) WT and (B)IL-9ko mice. (C) Survival plot of TUBO bearing mice, showing 100% ofIL-9ko mice surviving after day 85 post tumor injection (p<0.0001).Growth of 1×10⁶ 4T1 cells implanted s.c. in the flank of (D) WT and (E)IL-9ko mice. (F) Survival plot of 4T1 bearing mice, showing 75% ofIL-9ko mice surviving after day 50 post tumor injection (p<0.0001).Growth of 1×10⁵ CT26 cells implanted s.c. in the flank of (G) WT and (H)IL-9ko mice. (I) Survival plot of CT26 bearing mice, showing 75% ofIL-9ko mice surviving after day 50 post tumor injection (p<0.001).Arrows denote the day of rechallenge with 1×10⁶ TUBO or 4T1 cells (7 and8 IL-9ko mice respectively). Data are cumulative of a minimum of 2experiments.

FIG. 2. Spontaneous, autochthonous mammary tumors appear by four monthsof age in female mice BALB/neuT mice (WT). Double transgenic IL-9ko/neuTmice were generated, and the appearance of autochthonous tumors ascompared to WT/neuT mice was monitored. Autochthonous breast carcinomatumors appeared significantly later in female IL-9 deficient neuT micethan in WT/neuT mice p=0.002. This suggests that IL-9 may have a role intumor immunosurveillance.

FIGS. 3A-C. CD8⁺ T cells are essential for tumor rejection in IL-9komice. (A) Growth of 1×10⁶ 4T1 cells implanted s.c. in the flank ofIL9-ko mice treated with neutralizing antibodies against CD4, CD8,CD4⁺CD8 or isotype control. There were 6-8 mice per cohort and datashown are cumulative of two separate experiments. (B) Average tumorsizes in each treatment cohort 21 days post tumor injection. (C)Depletion of CD8⁺ T cells with anti-CD8 antibody in the IL-9ko mice thatrejected 4T1 tumors from 2A. Arrows indicate dosage times once weeklyfor three weeks.

FIGS. 4A-E. CD8⁺ T cells activated in a tumor specific manner and aretumor tropic. (A) ELISpot analysis measuring IFNγ⁺ spots derived fromtotal splenocytes (spleen) and lymphocytes (LN) from 4T1 bearing WT andIL-9ko mice. (B) Graph showing the average number of IFNγ⁺ spots fromtriplicate wells. *asterisk indicates p=0.001. (C) CD8⁺ T cells derivedfrom naïve or tumor bearing WT and IL-9ko mice co-cultured with either4T1 or BM185. Wells shown are representative of triplicatedeterminations from three biological replicates. (D) Graph showing theaverage numbers of IFNγ⁺ spots in each well. Biological replicates aredenoted as follows: WT naïve (WT N1-3), WT bearing 4T1 (WT TB1-3),IL-9ko naïve (IL9ko N1-3) and IL-9ko bearing 4T1 (IL9ko TB1-3). The*asterisk represents the significance of the difference in number ofspots when comparing CD8+ T cells from tumor bearing WT versus IL-9komice (p=0.007). Representative data of duplicate experiments with threemice in each condition. (E) Immunohistochemical evaluation of CD8⁺ Tcells present in 4T1 tumor derived from WT (lower panel) and IL-9ko(upper panel) mice. Each series is comprised of sequential slides toshow morphology (H&E), anti-CD8 staining, and a negative control. Imagesare representative of three tumors from three individual mice in eachstrain. Bar represents 100 μm. M=media, PMA=Phorbol 12-myristate13-acetate.

FIGS. 5A-B. Splenocytes, or CD8⁺ T cells from IL-9ko mice that rejected4T1 tumors, also impede 4T1 growth in WT mice. (A) Splenocytes derivedfrom tumor bearing WT (WT+4T1) or IL-9ko (IL-9ko+4T1) mice were mixedwith 4T1 cells and implanted s.c. in the flank of WT mice in the givenratios (100:1 and 33:1), holding the number of 4T1 cells constant at2.5×10⁴. Each line represents tumor growth in one mouse, and the cohortsare colored as follows: 1) 4T1 only, blue 2) IL-9ko+4T1 100:1, darkgreen 3) IL-9ko+4T1 33:1, light green 4) WT+4T1 100:1 purple and 5)WT+4T1 33:1, red. Shown is one of two determinations. (B) Enriched CD8+T cells derived from either naïve or 4T1 tumor bearing, WT or IL-9komice were co-injected s.c. into WT mice in a ratio of 25:1. Each cohortcontains duplicate mice injected with 4T1 and cells from threeindividual donor mice. Each line represents tumor growth in one mouse,and the cohort colors define the CD8+ T cell donor sub-sets asfollows: 1) WT+4T1, dark blue 2) WT naïve, light blue 3) IL-9ko+4T1,dark green and 4) IL-9ko naïve, light green.

FIGS. 6A-B. Anti-IL-9 treatment results in slowed tumor growth in WTmice. (A) Representation of the treatment schedule to measure the effectof IL-9 depletion on tumor growth. Symbols mark the days of anti-IL-9injections, twice a week for 3 weeks (3×). 2.5×10⁴ 4T1 cells wereinjected on day 0. (B) Box-whisker plot of tumor growth in 3 cohorts ofmice: untreated (white boxes, n=7), isotype control antibody (dottedboxes, n=8) and anti-IL-9 antibody (checkered boxes, n=11). Each boxcontains a line representing the median, and is bounded by the upper andlower quartiles. Minimum and maximum values are shown as whiskers. Thebar frames the period of time (days 0 to 15) during which there is asignificant difference in growth between the isotype control andanti-IL-9 treated cohorts (*asterisk, p=0.03). Data is cumulative of twoindependent experiments.

FIGS. 7A-B. IL-9 deficiency prevents experimental 4T1 metastasis growthin the lung, whereas addition of recombinant IL-9 eliminates theprotective quality of IL-9 deficiency. (A) 4T1 breast carcinoma cellswere injected in the tail vein of WT and IL-9ko mice. The number ofmacroscopic tumors from each lung are shown. Cross bars indicate themedian number of metastases, and bounding bars represent upper and lowerquartiles. (B) Representative images of 4T1 foci in lungs from eachtreatment group. IL-9 deficiency led to a significantly reduction innumber of lung lesions, suggesting that IL-9 contributes to the seedingor survival of circulating tumor cells.

DETAILED DESCRIPTION

This document provides methods and materials related to using inhibitorsof IL-9 signaling in conjunction with chemotherapy to treat cancer(e.g., solid tumors). For example, this document provides methods andmaterials for using inhibitors of IL-9 signaling (e.g., anti-IL9antibody preparations) in conjunction with chemotherapy to treat cancer(e.g., solid tumors such as breast cancer tumors or colon cancer tumors)or to reduce the growth rate of cancer (e.g., solid tumors such asbreast cancer tumors or colon cancer tumors) within a mammal. Examplesof inhibitors of IL-9 signaling include, without limitation, anti-IL9antibodies, anti-IL-9 receptor antibodies, siRNA molecules against IL-9,and siRNA molecules against an IL-9 receptor.

The term “antibody” as used herein refers to intact antibodies as wellas antibody fragments that retain some ability to bind an epitope. Suchfragments include, without limitation, Fab, F(ab′)2, and Fv antibodyfragments. The term “epitope” refers to an antigenic determinant on anantigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules (e.g., amino acid or sugar residues) and usually have specificthree dimensional structural characteristics as well as specific chargecharacteristics.

The antibodies provided herein can be any antibody (e.g., a monoclonalantibody) having binding affinity (e.g., specific binding affinity) forIL-9 or an IL-9 receptor. For example, an anti-IL-9 antibody preparationor an anti-IL-9 receptor antibody preparation provided herein can be apreparation of Fab fragments having the ability to bind to IL-9 (e.g.,human IL-9) or IL-9 receptor (e.g., a human IL-9 receptor). Anyappropriate method can be used to produce Fab fragments from intactantibodies. For example, standard papain digestion methods can be usedto make a Fab antibody preparation.

Antibodies provided herein can be prepared using any appropriate method.For example, a sample containing a human IL-9 polypeptide or human IL-9receptor polypeptide can be used as an immunogen to elicit an immuneresponse in an animal such that specific antibodies are produced. Theimmunogen used to immunize an animal can be chemically synthesized orderived from translated cDNA. In some cases, the immunogen can beconjugated to a carrier polypeptide, if desired. Commonly used carriersthat are chemically coupled to an immunizing polypeptide include,without limitation, keyhole limpet hemocyanin (KLH), thyroglobulin,bovine serum albumin (BSA), and tetanus toxoid.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, e.g., Green et al., Production of Polyclonal Antisera,in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1 5 (Humana Press 1992)and Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats,Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1(1992). In addition, those of skill in the art will know of varioustechniques common in the immunology arts for purification andconcentration of polyclonal antibodies, as well as monoclonal antibodies(Coligan, et al., Unit 9, Current Protocols in Immunology, WileyInterscience, 1994).

The preparation of monoclonal antibodies also is well-known to thoseskilled in the art. See, e.g., Kohler & Milstein, Nature 256:495 (1975);Coligan et al., sections 2.5.1 2.6.7; and Harlow et al., ANTIBODIES: ALABORATORY MANUAL, page 726 (Cold Spring Harbor Pub. 1988). Briefly,monoclonal antibodies can be obtained by injecting mice with acomposition comprising an antigen, verifying the presence of antibodyproduction by analyzing a serum sample, removing the spleen to obtain Blymphocytes, fusing the B lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures. Monoclonal antibodies can be isolated and purifiedfrom hybridoma cultures by a variety of well established techniques.Such isolation techniques include affinity chromatography with Protein ASepharose, size exclusion chromatography, and ion exchangechromatography. See, e.g., Coligan et al., sections 2.7.1 2.7.12 andsections 2.9.1 2.9.3; Barnes et al., Purification of Immunoglobulin G(IgG), in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79 104 (HumanaPress 1992).

In addition, methods of in vitro and in vivo multiplication ofmonoclonal antibodies are well known to those skilled in the art.Multiplication in vitro can be carried out in suitable culture mediasuch as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionallyreplenished by mammalian serum such as fetal calf serum, or traceelements and growth sustaining supplements such as normal mouseperitoneal exudate cells, spleen cells, and bone marrow macrophages.Production in vitro provides relatively pure antibody preparations andallows scale up to yield large amounts of the desired antibodies. Largescale hybridoma cultivation can be carried out by homogenous suspensionculture in an airlift reactor, in a continuous stirrer reactor, or inimmobilized or entrapped cell culture. Multiplication in vivo may becarried out by injecting cell clones into mammals histocompatible withthe parent cells (e.g., osyngeneic mice) to cause growth of antibodyproducing tumors. Optionally, the animals are primed with a hydrocarbon,especially oils such as pristane (tetramethylpentadecane) prior toinjection. After one to three weeks, the desired monoclonal antibody isrecovered from the body fluid of the animal.

In some cases, the antibodies provided herein can be made usingnon-human primates. General techniques for raising therapeuticallyuseful antibodies in baboons can be found, for example, in Goldenberg etal., International Patent Publication WO 91/11465 (1991) and Losman etal., Int. J. Cancer, 46:310 (1990).

In some cases, the antibodies can be humanized monoclonal antibodies.Humanized monoclonal antibodies can be produced by transferring mousecomplementarity determining regions (CDRs) from heavy and light variablechains of the mouse immunoglobulin into a human variable domain, andthen substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions when treating humans. Generaltechniques for cloning murine immunoglobulin variable domains aredescribed, for example, by Orlandi et al., Proc. Nat'l. Acad. Sci. USA86:3833 (1989). Techniques for producing humanized monoclonal antibodiesare described, for example, by Jones et al., Nature 321:522 (1986);Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science239:1534 (1988); Carter et al., Proc. Nat'l. Acad. Sci. USA 89:4285(1992); and Sandhu, Crit. Rev. Biotech. 12:437 (1992); Singer et al., J.Immunol. 150:2844 (1993). In some cases, humanization such as superhumanization can be used as described elsewhere (Hwang et al., Methods,36:35-42 (2005)). In some cases, SDR grafting (Kashmiri et al., Methods,36:25-34 (2005)), human string content optimization (Lazar et al., Mol.Immunol., 44:1986-1998 (2007)), framework shuffling (Dall'Acqua et al.,Methods, 36:43-60 (2005); and Damschroder et al., Mol. Immunol.,44:3049-3060 (2007)), and phage display approaches (Rosok et al., J.Biol. Chem., 271:22611-22618 (1996); Radar et al., Proc. Natl Acad. Sci.USA, 95:8910-8915 (1998); and Huse et al., Science, 246:1275-1281(1989)) can be used to obtain anti-IL-9 or anti-IL-9 receptor antibodypreparations. In some cases, fully human antibodies can be generatedfrom recombinant human antibody library screening techniques asdescribed elsewhere (Griffiths et al., EMBO 1, 13:3245-3260 (1994); andKnappik et al., J. Mol. Biol., 296:57-86 (2000)).

Antibodies provided herein can be derived from human antibody fragmentsisolated from a combinatorial immunoglobulin library. See, for example,Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2,page 119 (1991) and Winter et al., Ann. Rev. Immunol. 12: 433 (1994).Cloning and expression vectors that are useful for producing a humanimmunoglobulin phage library can be obtained, for example, fromSTRATAGENE Cloning Systems (La Jolla, Calif.).

In addition, antibodies provided herein can be derived from a humanmonoclonal antibody. Such antibodies can be obtained from transgenicmice that have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens and can be usedto produce human antibody secreting hybridomas. Methods for obtaininghuman antibodies from transgenic mice are described by Green et al.(Nature Genet., 7:13 (1994)), Lonberg et al. (Nature, 368:856 (1994)),and Taylor et al. (Int. Immunol., 6:579 (1994)).

Antibody fragments can be prepared by proteolytic hydrolysis of anintact antibody or by the expression of a nucleic acid encoding thefragment. Antibody fragments can be obtained by pepsin or papaindigestion of intact antibodies by conventional methods. For example, Fabfragments can be produced by enzymatic cleavage of antibodies withpapain. In some cases, antibody fragments can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′)2. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 3.5S Fab′monovalent fragments. In some cases, an enzymatic cleavage using pepsincan be used to produce two monovalent Fab′ fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg (U.S.Pat. Nos. 4,036,945 and 4,331,647). See also Nisonhoff et al., Arch.Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959);Edelman et al., METHODS IN ENZYMOLOGY, VOL. 1, page 422 (Academic Press1967); and Coligan et al. at sections 2.8.1 2.8.10 and 2.10.1 2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used provided the fragments retain some ability to bind (e.g.,selectively bind) its epitope.

The antibodies provided herein can be substantially pure. The term“substantially pure” as used herein with reference to an antibody meansthe antibody is substantially free of other polypeptides, lipids,carbohydrates, and nucleic acid with which it is naturally associated.Thus, a substantially pure antibody is any antibody that is removed fromits natural environment and is at least 60 percent pure. A substantiallypure antibody can be at least about 65, 70, 75, 80, 85, 90, 95, or 99percent pure.

As described herein, inhibitors of IL-9 signaling (e.g., anti-IL9antibody preparations) can be used in conjunction with chemotherapy totreat cancer or to reduce the growth rate of cancer within a mammal. Forexample, at least one inhibitor of IL-9 signaling can be administered toa mammal with breast or colon cancer before, together with, or followingthe administration of at least one chemotherapeutic agent. Examples ofcancers that can be treated using a combination of at least oneinhibitor of IL-9 signaling and at least one chemotherapeutic agentinclude, without limitation, breast cancers and colon cancers. Examplesof chemotherapeutic agents that can be used in combination with at leastone inhibitor of IL-9 signaling to treat cancer or to reduce the growthrate of cancer within a mammal as described herein include, withoutlimitation, anti-PD-1, anti-PD-L1, anti-CTLA4, Herceptin,cyclophosphamide, gemcitabine, capecitabine, azacytadine, bortezomib,carboplatin, cisplatin, etoposide, imatinib, 5-fluorouracil/leucovorin,docetaxel, paclitaxel, nab-paclitaxel, irinotecan, doxorubicin,methotrexate, and oxaliplatin therapies.

In some cases, inhibitors of IL-9 signaling (e.g., anti-IL9 antibodypreparations) can be used alone or in conjunction with at least onechemotherapeutic agent and/or at least one anti-cancer immunotherapeuticagent to treat cancer (e.g., solid tumors) or to reduce the growth rateof cancer (e.g., solid tumors) within a mammal. For example, at leastone inhibitor of IL-9 signaling can be administered to a mammal withbreast or colon cancer before, together with, or following theadministration of at least one anti-cancer immunotherapeutic agent.Examples of anti-cancer immunotherapeutic agents that can be used incombination with at least one inhibitor of IL-9 signaling to treatcancer or to reduce the growth rate of cancer within a mammal asdescribed herein include, without limitation, anti-cancer vaccines.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Inhibition of Adaptive Immunity by IL-9 can beDisrupted to Achieve Rapid T Cell Sensitization and Rejection ofProgressive Tumor Challenges

Mice, Cell Lines, and Reagents

BALB/c mice were purchased from NCI (Fredrick, Md.), and IL-9ko micewere obtained from Andrew McKenzie (MRC, Laboratory of MolecularBiology, Cambridge, UK). They were housed under specific pathogen-freeconditions. TUBO cells are derivatives of a spontaneous mammarycarcinoma in WT mice and were obtained from Dr. Forni (Torino, Italy).4T1 are a mammary carcinoma line derived from 410.4 mammary tumors. BM185 cells were derived from bone marrow from an acute lymphoblasticleukemia model, originally provided by D. Kohn (University of SouthernCalifornia, Los Angeles). All were maintained in complete RPMI 1640medium supplemented with 10% FCS, 2 mM glutamine, 5×10⁻⁵ M 2-ME, and 50μg/ml gentamicin.

Anti-IL-9 mAb (MM9C1) and its isotype control Ab were obtained from Dr.Jacques Van Snick (Ludwig Institute, Brussels, Belgium). Depletingantibodies were obtained as follows: anti-CD4 (clone GK 1.5, BioLegend),anti-CD8α (clone 2.43, LifeSpan BioSciences) antibodies or thecorresponding rat IgG isotype control. CD4⁺ and CD8⁺ T cells wereenriched using negative selection kits (Invitrogen, Carlsbad, Calif.).

In Vivo Tumor Studies

WT and IL-9ko mice were implanted subcutaneously (s.c.) with 1×10⁶ TUBO,or 1×10⁶ 4T1, measured twice weekly and sacrificed when the tumorsreached 1 cm² or showed signs of external necrosis. Tumor volume wascalculated using two perpendicular measurements in the followingformula: [a²xb/2]. For CD4⁺ and CD8⁺ T cell depletion experiments,anti-CD4 and -CD8 antibodies and the corresponding isotype control weredelivered i.p. at 125 μg each. Mice were pretreated 3-4 days beforetumor inoculation, and then subsequently once weekly for 4 weeks. ForIL-9 neutralization experiments, WT mice were injected s.c. with 2.5×10⁴4T1 cells and anti-IL-9, isotype control Ab (100 μg each) or leftuntreated. Abs were delivered to WT and IL-9ko mice throughintra-peritoneal (i.p.) injections three times per week for 3 weeks.

ELISpot Assays

1×10⁵ 4T1 splenocytes were co cultured with either 5×10⁴ 4T1 or TUBOcells. 5×10⁴ 4T1 lymphocytes were co cultured with either 2.5×10⁴ 4T1 orTUBO cells, following the manufacturers' instructions. CD4⁺ and CD8⁺ Tcells were enriched from the spleens of naïve and 4T1-bearing WT andIL-9ko mice using a negative isolation kit (Invitrogen, CarlsbadCalif.). 1×10⁵ T cells were co-cultured with 2.5×10⁴ 4T1 cells or withBM185, a non-specific control tumor line for 40 hours. Each experimentalcondition was executed in biological triplicates, which in turncomprised triplicate wells. Manufacturer's instructions were followed toreveal INFγ positive spots using the interferon gamma ELISpot kit fromMabtech (cat #3321-2HW-Plus). The plates were imaged and evaluated byZellNet Consulting, Inc. (Fort Lee N.J.), and result expressed asaverage of triplicate spots per condition. Phorbol 12-myristate13-acetate (PMA) was used as a positive control of cell activation.

Immunohistochemistry

4T1 tumors were harvested from IL-9ko and WT mice, formalin fixed, andparaffin embedded. Four micron sections were incubated overnight with orwithout anti-CD8 alpha antibody (Thermo scientific, MA5-16761)overnight, followed by detection using ImmPRESS™ Reagent Anti Rat IgG,peroxidase (Vector Labs MP-7404) followed by DAB substrate kit (Vectorlabs SK-4100). Slides were mounted in Permount and visualized with aLeica DMRB microscope. Images were acquired at a magnification of 200×with a numerical aperture of 2, at room temperature with an Olympus DP71camera using the DPController software (Olympus).

Winn Assays

Total splenocytes were harvested from 4T1 bearing WT, and from IL-9komice that had rejected 1×10⁶ 4T1 cells 2-3 months prior, and which wererechallenged with 5×10⁵ 4T1 cells one week before the start of theassay. Splenocytes were mixed with 2.5×10⁴ 4T1 cells and co-injecteds.c. into WT mice in the following proportions of splenocytes to 4T1cells: 100:1, 33:1 and only 4T1. Mice were monitored twice a week fortumor growth and tumor growth was compared to that of 4T1 cells mixedwith splenocytes derived from tumor bearing WT. A repeat experiment wasdone using negatively enriched CD8+T from tumor bearing and naïve WT andIL-9ko mice, cells were co-injected with 4T1 cells at a concentration of25:1 (CD8⁺ T cells:4T1 cells). Each experimental cohort consisted of Tcells isolated from three individual mice, and each individual isolatewas injected in duplicate, bringing the total per condition to six.

Statistical Analyses

Statistical significance of data was determined in most cases using theStudent's t-test to evaluate the p value. The log-rank (Mantel-Cox) testwas used to evaluate significant differences in survival. For thecomparison of differences in growth curves, the tumor size was comparedover days 9, 12, and 15 between groups using repeated measures analysisof variance.

Mammary Carcinomas TUBO and 4T1, as Well as Colon Carcinoma CT26 areRejected in IL-9ko Mice

IL-9ko mice were used to investigate the role of IL-9 in inhibitinganti-tumor immune activity. Subcutaneous (s.c.) injection of TUBO cellsin the flank of BALB/c (WT) mice resulted in robust tumor growth within10 days after tumor delivery (n=7, FIG. 1A). Similar s.c. tumorinoculations in IL-9ko mice had a markedly different effect: tumors wererejected in 78% of the IL-9ko mice (21/27 mice) (FIG. 1B). In the 6 micethat developed tumors, the average onset of macroscopic tumor growth wasdelayed to 60 days (FIG. 1B, p=0.0001). In addition to significantlydelayed tumor onset, slower growth was observed in IL-9ko mice,resulting in 100% survival 85 days after tumor delivery, compared to 0%survival at day 30 in the WT control group (FIG. 1C, p<0.0001). Toconfirm that tumor rejection was based on an immune component and notdue to a systemic effect linked to IL-9 deficiency, the TUBO challengewas repeated in 7 of the IL-9ko mice that rejected TUBO two months afterthe initial challenge (FIG. 1C, arrow). All the mice failed to developtumors, suggesting that these rechallenged mice developed a memoryresponse to TUBO cells.

This experiment was repeated with 4T1 cells, and palpable tumors weredetected 5 days post injection, which grew exponentially in WT mice(n=10, FIG. 1D). In contrast, 68% (15/22) of IL-9ko mice failed todevelop or rejected tumors (FIG. 1E). Of the remaining 32% (7/22) ofIL-9ko mice that developed tumors, tumor growth was significantly slowercompared to WT mice, and 75% of the IL-9ko mice survived past day 50post tumor inoculation (FIG. 1F, p<0.0001). To confirm that 4T1rejection also elicited a memory response, 8 of the IL-9ko mice thatrejected 4T1 were rechallenged with 4T1 (FIG. 1F, arrow). As before, allthe mice failed to develop tumors, suggesting the existence of a memoryresponse to 4T1 cells. None of the IL-9ko mice in which original 4T1tumors progressed exhibited any evidence of macrometastasis in thelungs, liver or spleen (evaluated 80-100 days post injection), which isroutinely observed in WT mice.

To investigate whether the tumor rejection seen in IL-9ko mice wasconfined to mammary carcinomas, the colon carcinoma cell line CT26 wasinjected into both WT (n=15) and IL-9ko mice (n=16). CT26 tumorsdeveloped in 87% (13/15) of WT mice (FIG. 1G), whereas tumors developedin only 25% (4/16) of IL-9ko mice injected (FIG. 1H). Once more, thetumors that developed in IL-9ko mice grew slower than those in WT mice,and 75% of IL-9ko mice remained tumor free 30 days after the initialchallenge (p=0.001). At this point, ten IL-9ko and two WT mice wererechallenged with CT26 (FIG. 1I, arrow). No tumor growth was observed inten IL-9ko mice and one WT mouse (CT26 tumor developed in the second WTmouse), again suggesting a memory response in IL-9ko mice.

IL-9 Deficiency Leads to Delayed Onset of Autochthonous Mammary Tumorsin Her2/Neu Transgenic Mice

Double transgenic mice deficient in IL-9 and expressing activatedHer2/neu were monitored from birth to track tumor onset and growth ascompared to that of BALB/c/Her2/neu mice. Mice were sacrificed when oneor more mammary tumors reached 10 mm², and their life span was recordedin weeks. A survival plot (FIG. 2) segregating males from females showeda significant increase in survival in females deficient in IL-9(p=0.01).

T Cells are Essential for Tumor Rejection in IL-9ko Mice

Evaluation of the immune composition of spleens and lymph nodes of 4T1bearing WT and IL-9ko mice revealed increased total numbers of CD4⁺ andCD8⁺ T cells in IL-9ko mice, and a concomitant decrease in numbers ofCD11b⁺Gr1⁺ cells. However, closer scrutiny revealed that any differencein total numbers was directly related to tumor size and not to IL-9status. Since tumor rejection occurred 10-14 days after tumorinoculation (FIG. 1E), timing that is reminiscent of an adaptive immuneresponse, and that this resulted in a memory response, whether T cellswere involved in tumor rejection in an IL-9 deficient context wasevaluated. To test this, IL-9ko mice were inoculated with 4T1 tumors,and CD4⁺ and CD8⁺ T cells were depleted with mAb. Cohorts comprised 6-8mice, and were treated as follows: anti-CD4, anti-CD8, both anti-CD4 andanti-CD8, isotype control antibody or untreated. Growth was monitoredover 30 days. Both the untreated and isotype control antibody treatedIL-9ko mice gave evidence of tumor rejection between days 10 and 14 posttumor injection, with rejection completed one week later. In contrast,CD8 depletion or joint CD4 and CD8 depletion resulted in 4T1 tumorgrowth indistinguishable from that observed in WT mice (FIG. 3A).Finally, depletion of only CD4⁺ T cells also prevented 4T1 rejection,but with slower tumor outgrowth than CD8⁺ depletion. These resultsdemonstrate that in an IL-9 deficient milieu, both CD8⁺ and CD4⁺ T cellswere involved in tumor eradication, and that neither subset alone wassufficient for cure. Tumor sizes were tabulated 21 days post tumorinjection, and confirm that CD8⁺ depletion resulted in large tumor(averaging 700 mm³) whereas untreated or isotype treated mice harboredvery small tumors (averaging 14 mm³) or no tumors at all (FIG. 3B).

These results suggest that the presence of IL-9 negatively regulates Tcell function within 10-14 days of tumor challenge. The precise sourceof IL-9 in the tumor microenvironment is yet to be determined. However,both 4T1 and TUBO cells do not transcribe either IL-9 or IL9R mRNA, nordo they secrete IL-9 in vitro, as measured by ELISA using supernatantsof tumors generated in IL-9ko mice and cultured for 3 days.

To test whether the absence of recurrent tumors in IL9ko 4T1 challengedmice reflected active T cell immunosurveillance, CD8⁺ T cells weredepleted in IL-9ko mice (n=8) that had rejected 4T1 cells 21 days prior(FIG. 3C, arrows). Tumors grew in 3/8 mice, and no tumor growth wasevident in 5/8 mice. These data confirmed that active T cellsurveillance was operative in at least a portion of non-recurrences, andraised the possibility that mice without tumor growth following CD8depletion might harbor sufficient CD4⁺ T cell immunologic memory tocompensate for the CD8 depletion, or that the tumor was completelyeradicated.

T Cells from IL-9ko Mice are Activated and Tumor Specific

To confirm that tumor rejection was due to the sensitization of a tumorspecific immune component, total splenocytes and lymphocytes (from tumordraining lymph nodes) were harvested from 4T1 bearing WT and IL-9ko mice14 days after tumor injection. These cells were co-cultured with either4T1 (target tumor) or TUBO (negative control tumor) cells to measure thenumber of cells that were activated in a tumor specific manner, usingthe number of IFNγ⁺ spots as a reporter of activation (FIG. 4A). Thenumber of IFNγ⁺ spots present in WT co-cultured with 4T1 cells was nearthe levels of negative control (TUBO) tumor. In contrast, IL-9ko derivedsplenocytes and lymphocytes were activated in a tumor specific manner.Moreover, IL-9ko derived splenocytes (6.5 fold increase) and lymphocytes(9.7 fold increase) showed a significantly higher degree of activationin the presence of 4T1 as compared to WT cells (FIG. 4B, p=0.001).

Since it was observed that T cell depletion resulted in tumor growth inIL-9ko mice, the ELISpot assay was repeated with isolated CD4⁺ T cellsand CD8⁺ T cells. These cells were co-cultured with 4T1 or BM185 cells,a BALB/c background murine leukemia cell line used here as a negativecontrol (FIG. 4C). Again using the number of IFNγ⁺ spots as a reporterof T cell activation, a 3.6 fold increase in the number of activatedCD8⁺ T cells was found in the population derived from tumor bearingIL-9ko mice as compared to their WT counterparts: an average of 239 ofIFNγ⁺ spots in cells from IL-9ko mice, versus 67 spots in WT cells (FIG.4D, p=0.007). Furthermore, activation of CD8⁺ T cells was 4T1 specific,since there were no measurable IFNγ⁺ spots when CD8⁺ T cells wereco-cultured with BM185 cells. Phorbol 12-myristate 13-acetate (PMA) wasused as a positive control to confirm that the WT CD8⁺ T cells werecapable of activation. A similar experiment using CD8⁺ T cells from TUBObearing mice yielded similar results. CD4⁺ T cells tested underidentical conditions (T cells plus tumor cell lines in the absence ofsyngeneic antigen-presenting cells) produced IFNγ⁺ spots only if exposedto PMA, consistent with an inability to recognize MHC Class IInon-expressing tumor lines directly.

CD8⁺ T Cells are Found in 4T1 Tumors Growing in IL-9ko Mice, but not inTumors Growing in WT Mice

Having observed that CD8⁺ T cells were key effectors in tumoreradication, and that they were activated in a tumor specific manner, itwas sought to verify the presence of CD8⁺ T cells in 4T1 tumors thatwere in the process of being rejected. WT and IL-9ko mice were injectedwith 4T1 cells, and tumor growth monitored. After 7 days, the tumors inWT mice were robustly growing (an average of 5 mm²) (FIG. 1D), whereastumors growing in IL-9ko mice were decreasing in size (2-3 mm²) (FIG.1E). Tumors were harvested at this point, formalin fixed, and paraffinembedded. Staining with anti-CD8 revealed a population of CD8⁺ cellsarranged mostly in groups at the margins of shrinking 4T1 tumors in theIL-9ko mice (FIG. 4E). No CD8⁺ cells were observed in tumors growing inWT mice, even though the ELISpot analyses revealed 4T1-specific CD8⁺ Tcells in the spleens of WT 4T1-bearing mice (FIG. 4D).

Splenocytes, or CD8⁺ T Cells from IL-9ko Mice that Rejected 4T1 Tumors,Also Abrogate 4T1 Growth in WT Mice

Winn assays were employed to test whether activated splenocytes fromIL-9ko mice that had rejected 4T1 tumors were capable of inducing tumorrejection in WT mice. IL-9ko mice that rejected 4T1 tumors wererechallenged with 4T1 cells to boost the levels of memory cells. Totalsplenocytes were then harvested from the rechallenged mice, and mixedwith 4T1 cells prior to injection in the flank of WT mice. WT mice weresegregated into 3 cohorts, which received increasing numbers ofsplenocytes, holding the number of tumor cells constant: 1) nosplenocytes added, 2) 33:1, and 3) 100:1. Splenocytes from 4T1 bearingWT mice were used in the same proportions as a control. Tumor growth wasmonitored twice weekly, revealing tumor growth in WT mice that receivedonly 4T1, and complete abrogation of tumor growth in all the mice thatreceived IL-9ko activated splenocytes (FIG. 5A). Co-injection of 4T1 andsplenocytes derived from 4T1 bearing WT mice grew similarly to the micethat received only 4T1. These results revealed that splenocytes derivedfrom mice that had rejected 4T1 contained cells that were capable oferadicating 4T1 tumor cells in a WT context.

The experiment was repeated with negatively isolated CD8⁺ T cells toverify the finding that depletion of CD8⁺ T cells enables tumor growthin IL-9ko mice (FIG. 3A). CD8⁺ T cells were harvested from spleens ofthe following groups of mice: 1) WT bearing 4T1 tumors, 2) naïve WT, 3)IL-9ko bearing 4T1 tumors, and 4) naïve IL-9ko. CD8⁺ T cells were mixedwith 4T1 cells in a ratio of 25:1, injected into WT mice, and tumorgrowth was monitored. As observed before (FIG. 5A), cells derived fromtumor bearing IL-9ko mice prevented 4T1 growth in 6/6 mice. Cellsisolated from both IL-9ko and WT naïve mice permitted 4T1 growth.Surprisingly, half of the mice treated with CD8⁺ T cells from tumorbearing WT mice prevented tumor growth, and half enabled tumor growth.These findings confirm the observations from the ELISpot assays (FIG.3C), which revealed that CD8⁺ T cells derived from IL-9ko mice have 3.6fold more tumor reactive cells than their WT equivalents.

Anti-IL-9 Treatment Results in Slowed Tumor Growth in WT Mice

If IL-9 is an important factor in tumor development, then neutralizingIL-9 in WT mice with nascent 4T1 should lead to slowed tumor growth ortumor rejection. WT mice were inoculated with 4T1 cells and separatedinto three cohorts: 1) untreated, 2) treated with neutralizinganti-IL-9, and 3) treated with isotype control (FIG. 6A), and tumorgrowth was monitored. Mice treated with anti-IL-9 antibody exhibitedsignificant delay in tumor growth (days 0-15) as compared to untreatedmice (p<0.0001), and also when compared to isotype control antibody(FIG. 6B, p=0.03). The difference in growth between anti-IL-9 anduntreated remained highly significant throughout the three weeks(p<0.0001). However, due to the high degree of variation in the isotypecontrol cohort there was no measurable significance in 4T1 growth inlatter time points.

IL-9 Deficiency Prevents the Establishment of 4T1 Tumor Foci in the Lung

Since few IL-9ko mice that developed 4T1 tumors never had evidence ofmacro metastases in the lungs, an experimental metastasis model was usedto verify whether IL-9 plays a role in 4T1 seeding in the lung. Tailvein injections of 1×10⁵ 4T1 cells led to metastatic lesions in WT mice(an average of 87 foci per lung), whereas the majority of IL-9ko micedid not develop visible metastases (an average of 3 foci per lung,p=0.0001) (FIGS. 7A and 7B). Addition of recombinant IL-9 led toenhanced 4T1 seeding in the lungs of IL-9ko mice (an average of 13 fociper lung) (p=0.043, FIGS. 7A and 7B).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

The invention claimed is:
 1. A method for treating a mammal having colonor breast cancer, wherein said method comprises administering atherapeutically effective amount of an inhibitor of IL-9 signaling aloneto said mammal, wherein said inhibitor is an anti-IL-9 antibody, andwherein the progression of said colon or breast cancer or the number ofcolon or breast cancer cells within said mammal is reduced.
 2. Themethod of claim 1, wherein said mammal is a human.